Electrophoresis 2D gel

The beauty of 2D gel electrophoresis as a separation technique is the orthogonality of the two separation dimensions separation by charge (isoelectric point) in the first dimension, and separation by size in the second dimension. Two-dimensional gel electropheresis is the core separation technique for proteomics, along with HPLC (for preparative isolation). 

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

Figure 2.2. Fractionation of protein extracts before 2D gel electrophoresis. Crude lysates can be fractionated by affinity purification or by a number of chromatographic techniques. In addition, organelles or other cellular structures can be purified and lysates from these organelles can be fractionated or separated directly on 2D gels. By repeating this procedure using a number of conditions it may be possible to visualize a large fraction of a cell s proteome.

Figure 2.4. Peptide fingerprinting by MALDI-TOF mass Spectrometry. Proteins are extracted and separated on by 2D gel electrophoresis. A spot of interest is excised from the gel, digested with trypsin, and ionized by MALDI. The precise mass of proteolytic fragments is determined by time-of- flight mass spectrometry. The identity of the protein is determined by comparing the peptide masses with a list of peptide masses generated by a simulated digestion of all of the open reading frames of the organism.

Current proteomics studies rely almost exclusively on 2D gel electrophoresis to resolve proteins before MALDI-TOF or ESI-MS/MS approaches. A drawback of the 2D gel approach is that it is relatively slow and work intensive. In addition, the in-gel proteolytic digestion of spots followed by mass spectrometry is a one-at-a-time method that is not well suited for high throughput studies. Therefore, considerable effort is being directed towards alternate methods for higher throughput protein characterization. 

Protein expression mapping by 2D gel electrophoresis and mass spectrometry... 

The use of 2D gel electrophoresis and mass spectrometry to identify proteins was discussed in Chapter 2. Protein expression mapping involves the use of these methodologies to compare expression patterns in different cell types or in the same cell type that has been exposed to different... 

The utility of protein expression mapping using 2D gel electrophoresis and mass spectrometry has been demonstrated for several experimental systems. One application has been to assess the differences in protein expression between normal and cancerous cells. For example, expression mapping has been used to identify protein markers for bladder cancer (Ostergaard et al., 1999). This was accomplished by identifying proteins released into the urine of patients with and without bladder cancer using 2D electrophoresis and mass spectrometry. 

Figure 3.1. Protein expression mapping using 2-D electrophoresis and mass spectrometry. The purpose is to compare protein expression patterns between cell types or in the same cell type under different growth conditions. Proteins are extracted from the different cell types and separated by 2D gel electrophoresis. Image analysis programs are used to compare the spot intensities between gels and identify proteins that are differentially expressed. The protein of interest is excised from the gel and its identity is determined by mass spectrometry. The power of the method increases greatly if the identity of a large number of proteins on the gel is known and present in a database because information can then be obtained without further mass spectrometry.

Figure 3.2. Stable isotope labeling for quantifying differential protein expression. Cell populations are grown in either 14N or 15N containing medium. Protein lysates are fractionated and separated by 2D gel electrophoresis. Protein spots are excised, digested with trypsin and the mass of the resulting peptides is determined by mass spectrometry. The presence of 15N results in a shift and creates two peaks for each peptide. The ratio of intensities of the peaks is indicative of the relative expression levels of the proteins. Spot A contains a protein that is expressed at similar levels in both cell pools. Spot B contains a protein that is expressed at higher levels in cell pool 2. Figure adapted from Oda et al. (1999).

Proteomics is an interdisciplinary science that includes biology, bioinformatics, and protein chemistry. The purpose of this book is to provide the reader with an overview of the types of questions being addressed in proteomics studies and the technologies used to address those questions. The first chapter is a concise outline of the field as it presently stands. The second chapter provides an overview of the use of 2D-gel electrophoresis and mass spectrometry to identify proteins, as well as post-translational... 

FIGURE 1.1 A 2D-gel electrophoresis map of colorectal epithelia cells proteins from the SWISS- 2DPage database (entry CATD HUMAN, primary access number P07339) accessible from http //www.expasy.org/swiss-2dpage. 

Stanislaus, R., Jiang, L.H., Swartz, M., Arthur, J. Almeida, J.S. (2004). An XML standard for the dissemination of annotated 2D gel electrophoresis data complemented with mass spectrometry results. BMC Bioinformatics 5, 9. 

First Dimension Optimization After the second-dimension separation has been developed, the first-dimension flow rate is determined. This includes selecting a first-dimension column diameter to work at the flow rate selected. We illustrate the selection process with an application that addresses a column method for proteins that functions as a replacement for planar 2D gel electrophoresis (2DGE) within a narrow molecular weight and p/range. In the planar experiment, isoelectric focusing is performed in the first dimension and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS/PAGE) in the second dimension. 

One of the primary challenges facing the field of separation science is the analysis of the entire complement of proteins produced by an organism—a field of research known as proteomics. 2D gel electrophoresis remains the gold standard in protein separations, with the ability to resolve as many as 5000 proteins in a single gel... 

Traditional methodologies such as 2D-gel electrophoresis and mass spectrometry have been considerably improved to resolve thousands of proteins in a single experiment. However, these approaches are both time consuming and unsuitable for the... 

Precise quantifications are an important quality in molecular biology. There are slight differences in the methods used for global and targeted proteomics. In experiments intended to visualize as many proteins as possible, it is highly desirable to have a parallel quantification method that builds on the display technique. For 2D gel electrophoresis, fluorescent staining methods are under development (Urwin and Jackson, 1993), but they still lack overall sensitivity. Labeling proteins with radioactive isotopes is the most precise method for quantification but is limited to cell cultures, and alternatives are desirable. Recently, a precise method... 

Fig. 1.43 Strategies for protein identification. (A) 2D gel electrophoresis approach. (B) 2D liquid chromatography approach. lEF Isoelectric focusing, sex strong cation exchange column, RP reverse phase column, SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis.

A number of molecules in groups 2 and 3 have been identified by the differential homing capacity of phage display libraries and combination peptide libraries [71]. Biochemical strategies such as the application of 2D gel electrophoresis on protein extracts from endothelial cell surfaces have also proven useful in this respect [72]. 

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